1. Field of the Invention
This invention relates to the use of recombinant-DNA technology and the use of Gram negative bacteria such as Escherichia coli (hereinafter called E.coli) to express and export gene products to the growth media of such organisms.
Thus, the invention provides for a process for expressing proteins in Gram negative bacteria to provide for extracellular secretion thereof. The invention furthermore provides for a recombinant DNA construct and plasmid vectors and Gram negative bacteria comprising such recombinant DNA construction. The invention also extends to proteins obtained by the process of the invention.
2. Description of the Related Art
The relatively new recombinant DNA technology, whereby novel recombinant DNA structures may be constructed and introduced into a prokaryotic or eukaryotic host cell, has made it theoretically possible to produce a great number of proteins. The use of bacteria to produce heterologous gene products has made it possible to obtain proteins which can otherwise only be obtained from natural sources at a considerable cost. Well known examples of polypeptides originally from humans produced in bacteria are human Growth Hormone (hGH), insulin, .alpha.-interferon, .gamma.-interferon, somatostatin and somatomedins (Insulin-like Growth Factors).
The use of bacteria to express foreign genes have faced many practical and biological problems including stability of the polypeptide due to proteolysis, level of expression, precipitation of the protein product correlated to misfolding and lack of biological activity of the protein after purification. To solve these problems a variety of techniques have been developed to be able to use the well characterized entero bacteria E.coli to express any gene product. These methods include the use of different promoters to be able to regulate the level of expression, gene fusions to stabilize normally unstable proteins in the cell and the use of signal peptides to translocate proteins out from the cytoplasm to the periplasmic space in where disulphide bridges can be formed in contrast to the cytoplasm where the reducing environment makes this formation difficult. Proteins with cystein bridges in the structure expressed in the cytoplasm of E.coli will usually not get the correct tertiary structure due to the difficulties associated with forming these bridges. This could potentially lead to precipitation of the polypeptide upon overproduction, a rapid proteolytic degradation if not precipitated in the cell and no biological activity of the expressed and purified polypeptide. This has been observed in E.coli for expression of proinsulin, insulin A-chain, insulin B-chain, Insulin-like Growth Factors and tissue specific Plasminogen Activator (t-PA). To overcome this problem the polypeptide has to be renatured after purification or secreted to the periplasmic space of E.coli where the correct folding can be potentially achieved. One other aspect of bacterial gene expression and secretion apart from folding and stability is the use of Gram positive bacteria. These organisms have a different organization of the membrane structures surrounding the cell cytoplasm compared to the Gram negative counterpart. The Gram positive prokaryotes have only one cell membrane and secreted proteins get exported to the growth medium where secretion in the Gram negative E.coli locates the protein to the periplasmic space due to a double membrane layer surrounding the cell cytoplasm. By using Gram positive bacteria, secreted gene products can be collected from the growth media which would facilitate the downstream processing of the product. The emphasis to use Gram positive bacteria in industrial processes is thus correlated to this secretory process, but from other aspects it would be preferred to use the well characterized E.coli in large scale production of gene products simply because expression systems are more developed for this organism.